Role of the conserved glutamine 291 in the rat γ-aminobutyric acid transporter rGAT-1

Abstract

Abstract. We investigated the role of the Q291 glutamine residue in the functioning of the rat g-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transportcoupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wildtype and Q291 mutants suggest that GAT-1 is a functional monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating the impact of impermeant sulfhyd- Cell. Mol. Life Sci. 63 (2006) 0100-0111 1420-682X/06/010100-12 DOI 10.1007/s00018-005-5512-6 © Birkhäuser Verlag, Basel, 2006 ryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium or in maintaining the cation accessibility to the transporter. Key words. Neurotransmitter transporter; GAT-1; electrophysiology; site-directed mutagenesis; Xenopus laevis oocyte; structure-function relationship.g-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in mammalian brain, and the GABA transporter GAT-1 is an integral membrane protein responsible for the reuptake of GABA from the synaptic cleft during neurotransmission. GAT-1 was the first cloned member of a large family of homologous proteins, the Na + /Cl --dependent neurotransmitter transporters [1], which includes transporters for other neurotransmitters such as norepinephrine, dopamine, serotonin and glycine, as well as for a number of other substrates that share the common property of being amino acids or amino acid derivatives [2]. These membrane proteins are predicted to have 12 transmembrane-spanning domains (TMDs) linked by hydrophilic loops with the NH 2 and COOH terminals inside the cell, a topological model that has been confirmed for the serotonin transporter by means of sitedirected chemical labeling [3]. GAT-1 mediates the electrogenic reuptake of GABA in the presence of sodium and chloride ions by means of a mechanism that has been extensively studied [4][5][6][7][8][9][10][11][12]. Many attempts have been made to determine which of the transporter amino acid residues are involved in the interaction with GABA and with Na + and Cl -ions, and which participate in the conformational changes associated with translocation [13][14][15]. As GABA is a zwitterionic molecule and its cosubstrates are charged species, the first studies focused on the charged and conserved residues predicted to be located in (or adjacent to) the transmembrane (TM) domains. This allowed the identification of * Corresponding author. Cellular and Molecular Life Sciences Research ArticleRole of the conserved glutamine 291 in the r...

A Glutamine Residue Conserved in the Neurotransmitter:Sodium:Symporters Is Essential for the Interaction of Chloride with the GABA Transporter GAT-1

“…2, gray bars), in full agreement with an earlier study (20). This loss of activity is not due to decreased steady-state levels of the mutant transporters on the plasma membrane relative to WT, because the intensity of the bands in the biotinylated fraction of these and almost all of the other mutants studied is similar to that of WT as shown by surface biotinylation (Table 1 and supplemental Fig.…”

“…Because several of the homologous residues are critical for transport in GAT-1 (16 -19), it is likely that the formation of the thin gate is an obligate intermediate during substrate translocation: the transition from the outward-facing conformation to the inward-facing conformation. A highly conserved glutamine residue, Gln-291 in GAT-1 (Gln-250 in LeuT), has been reported to be crucial for GABA transport by GAT-1 (20). Inspection of the LeuT structure and the putative chloride-binding site suggests two possible reasons why this residue is crucial for transport: 1) hydrogen bond formation with the conserved arginine residue; Arg-69 in GAT-1 (Arg-30 in LeuT), is critical for the formation of the thin gate; and 2) direct interaction with the cotransported chloride predicted by one model (5) (Fig.…”

Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1

“…Figure 10 also shows that the reducing agent DTT (12 mM), after treatment with 0.2 mM CuPh, significantly restored the activity of the K102C/D338C mutant. Recent studies have indicated that members of the NSS family form constitutive oligomers (12,13,15,17,19,20,31,32), although the physiological meaning of this process is still under investigation (17,25,33,35,37). Since KAAT1 belongs to the NSS family, we also considered the hypothesis that CuPh may actually cause the creation of a disulfide bond between two cysteines in different subunits of a KAAT1 homo-oligomer.…”

Functional Defects in the External and Internal Thin Gates of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Can Compensate Each Other

“…eral of the homologous residues are critical for transport in GAT-1 (12)(13)(14)(15)(16)(17), it is likely that the formation of the thin gate is an obligate intermediate during substrate translocation: the transition from the outward-to the inward-facing conformation. We have recently shown that perturbation of this thin gate of GAT-1 by mutation increases the proportion of outwardfacing transporters (6).…”